Three Dimensional Carbon Nanosheets as a Novel Catalyst Support for Enzymatic Bioelectrodes

A high performance laccase‐based biofuel cell cathode is developed using carbon nanosheets (CNS) as the catalyst support and buckypaper (BP) as the substrate electrode. Compared to multiwalled carbon nanotube (MWNT)‐based electrodes, CNS‐based electrodes exhibit better electrochemical properties for the oxygen reduction reaction (ORR) under biologically relevant conditions. It is shown that CNSs are conformally coated on the nanotubule bundles within the BP and that laccase is intimately attached to the CNS‐BP. Electrochemical characterization is carried out to derive the kinetic parameters of the ORR at the laccase‐CNS‐BP cathode. The laccase‐CNS‐BP exhibits a steep ORR cathodic wave with a Tafel slope of 19 mV decade^‐1. The onset potential obtained for laccase ORR at CNS‐BP is 20 mV higher than that of the MWNT‐based electrodes, and the laccase‐CNS‐BP cathode has a higher current density than MWNT electrodes.     

Oxygen-reducing enzyme cathodes produced from SLAC, a small laccase from Streptomyces coelicolor

The bacterially-expressed laccase, small laccase (SLAC) of Streptomyces coelicolor, was incorporated into electrodes of both direct electron transfer (DET) and mediated electron transfer (MET) designs for application in biofuel cells. Using the DET design, enzyme redox kinetics were directly observable using cyclic voltammetry, and a redox potential of 0.43 V (SHE) was observed. When mediated by an osmium redox polymer, the oxygen-reducing cathode retained maximum activity at pH 7, producing 1.5 mA/cm2 in a planar configuration at 900 rpm and 40 degrees C, thus outperforming enzyme electrodes produced using laccase from fungal Trametes versicolor (0.2 mA/cm2) under similar conditions. This improvement is directly attributable to differences in the kinetics of SLAC and fungal laccases. Maximum stability of the mediated SLAC electrode was observed at pH above the enzyme's relatively high isoelectric point, where the anionic enzyme molecules could form an electrostatic adduct with the cationic mediator. Porous composite SLAC electrodes with increased surface area produced a current density of 6.25 mA/cm2 at 0.3 V (SHE) under the above conditions.     

Molecular design of laccase cathode for direct electron transfer in a biofuel cell

In order to improve the direct electron transfer in enzymatic biofuel cells, a rational design of a laccase electrode is presented. Graphite electrodes were functionalized with 4-[2-aminoethyl] benzoic acid hydrochloride (AEBA). The benzoic acid moiety of AEBA interacts with the laccase T1 site as ligand with an association constant (K(A)) of 6.6×10(-6) M. The rational of this work was to orientate the covalent coupling of laccase molecule with the electrode surface through the T1 site and thus induce the direct electron transfer between the T1 site and the graphite electrode surface. Direct electron transfer of laccase was successfully achieved, and the semi-enzymatic fuel cell Zn-AEBA laccase showed a current density of 2977 μA cm(-2) and a power density of 1190 μW cm(-2) at 0.41 V. The molecular oriented laccase cathode showed 37% higher power density and 43% higher current density than randomly bound laccase cathode. Chronoaperometric measurements of the Zn-AEBA fuel cell showed functionality on 6 h. Thus, the orientation of the enzyme molecules improves the electron transfer and optimizes enzyme-based fuel cells efficiency.     

A mediated glucose/oxygen enzymatic fuel cell based on printed carbon inks containing aldose dehydrogenase and laccase as anode and cathode

Enzyme electrodes show great potential for many applications, as biosensors and more recently as anodes and cathodes in biocatalytic fuel cells for power generation. Enzymes have advantages over metal catalysts, as they provide high specificity and reaction rates, while operating under mild conditions. Here we report on studies related to development of mass-producible, completely enzymatic printed glucose/oxygen biofuel cells. The cells are based on filter paper coated with conducting carbon inks containing mediators and laccase, for reduction of oxygen, or aldose dehydrogenase, for oxidation of glucose. Mediator performance in these printed formats is compared to relative rate constants for the enzyme-mediator reaction in solution, for a range of anode and cathode mediators. The power output and stability of fuels cells using an acidophilic laccase isolated from Trametes hirsuta is greater, at pH 5, than that for cells based on Melanocarpus albomyces laccase, that shows optimal activity closer to neutral pH, at pH 6. Highest power output, although of limited stability, was observed for ThL/ABTS cathodes, providing a maximum power density of 3.5 μWcm(-2) at 0.34 V, when coupled to an ALDH glucose anode mediated by an osmium complex. The stability of cell voltage above a threshold of 200 mV under a moderate 75 kΩ load is used to benchmark printed fuel cell performance. Highest stability was obtained for a printed fuel cell using osmium complexes as mediators of glucose oxidation by aldose dehydrogenase, and oxygen reduction by T. hirsuta laccase, maintaining cell voltage above 200 mV for 137 h at pH 5. These results provide promising directions for further development of mass-producible, completely enzymatic, printed biofuel cells.     

Ultra‐Lightweight 3D Carbon Current Collectors: Constructing All‐Carbon Electrodes for Stable and High Energy Density Dual‐Ion Batteries

Dual‐ion batteries (DIBs) attract great interest because they allow two types of ions for reversibly intercalating into electrodes, resulting in various advantages. However, there are three critical problems using graphite‐based cathodes, namely, low active material proportion in the electrodes, current collector corrosion, and massive cathode variation. For addressing these problems, an ultra‐lightweight 3D carbon current collector (CCC) is developed to fabricate all‐carbon electrodes as both cathodes and anodes. Compared with the conventional DIBs using Al and Cu foils as current collectors, the DIBs with 3D CCC of electrically conductive pathways and sufficient ionic diffusion channels deliver enhanced specific capacity stabilized around 140 and 120 mAh g^?1 at 0.5 and 1C, respectively. The electrochemically inert 3D CCC could essentially promote the energy density when calculating the entire electrode mass, along with long‐life cycle stability of 1000 cycles at 5C and no electrochemical corrosion on either anodes or cathodes. With an in situ optical microscope, the cathode expansion is found to massively reduce because the porous 3D CCC could effectively alleviate the huge volume. The results suggest a novel strategy for achieving low‐cost and high energy density DIBs with both mechanically and electrochemically stable features.     

Mediatorless sugar/oxygen enzymatic fuel cells based on gold nanoparticle-modified electrodes

We report on the fabrication and characterisation of a gold-nanoparticle (AuNP)-based mediatorless sugar/oxygen biofuel cell (BFC) operating in neutral sugar-containing buffers and human physiological fluids, such as blood and plasma. First, Corynascus thermophilus cellobiose dehydrogenase (CtCDH) and Myrothecium verrucaria bilirubin oxidase (MvBOx), used as anodic and cathodic bioelements, respectively, were immobilised on gold electrodes modified with 20 nm AuNPs. Detailed characterisation and optimisation of a new CDH/AuNP-based bioanode were performed and the following fundamental parameters were obtained: (i) the redox potential of the haem-containing centre of the enzyme was measured to be 75 mV vs. NHE, (ii) the surface coverage of CtCDH was found to be 0.65 pmol cm(-2) corresponding to a sub-monolayer coverage of the thiol-modified AuNPs by the enzyme, (iii) a turnover number for CtCDH immobilised on thiol-modified AuNPs was calculated to be ca. 0.5 s(-1), and (iv) the maximal current densities as high as 40 μA cm(-2) were registered in sugar-containing neutral buffers. Second, both biomodified electrodes, namely the CtCDH/AuNP-based bioanode and the MvBOx/AuNP-based biocathode, were combined into a functional BFC and the designed biodevices were carefully investigated. The following characteristics of the mediator-, separator- and membrane-less, miniature BFC were obtained: in phosphate buffer; an open-circuit voltage of 0.68 V, a maximum power density of 15 μW cm(-2) at a cell voltage of 0.52 V and in human blood; an open-circuit voltage of 0.65 V, a maximum power density of 3 μW cm(-2) at a cell voltage of 0.45 V, respectively. The estimated half-lives of the biodevices were found to be >12, <8, and <2 h in a sugar-containing buffer, human plasma, and blood, respectively. The basic characteristics of mediatorless sugar/oxygen BFCs were significantly improved compared with previously designed biodevices, because of the usage of three-dimensional AuNP-modified electrodes.     

Design of Carbon Nanotube‐Based Gas‐Diffusion Cathode for O2 Reduction by Multicopper Oxidases

Multicopper oxidases, such as laccase or bilirubin oxidase, are known to reduce molecular oxygen at very high redox potentials, which makes them attractive biocatalysts for enzymatic cathodes in biological fuel cells. By designing an enzymatic gas‐diffusion electrode, molecular oxygen can be supplied through the gaseous phase, avoiding solubility and diffusion limitations typically associated with liquid electrolytes. In doing so, the current density of enzymatic cathodes can theoretically be enhanced. This publication presents a material study of carbon/Teflon composites that aim to optimize the functionality of the gas‐diffusion and catalytic layers for application in enzymatic systems. The modification of the catalytic layer with multiwalled carbon nanotubes, for example, creates the basis for stronger π–π stacking interactions through tethered enzymatic linkers, such as pyrenes or perylene derivates. Cyclic voltammograms show the effective direct electron contact of laccase with carbon nanotube‐modified electrodes via tethered crosslinking molecules as a model system. The polarization behavior of laccase‐modified gas‐diffusion electrodes reveals open‐circuit potentials of +550 mV (versus Ag/AgCl) and current densities approaching 0.5 mA cm² (at zero potential) in air‐breathing mode.     

A biofuel cell with enhanced performance by multilayer biocatalyst immobilized on highly ordered macroporous electrode

A one-compartment glucose/O(2) biofuel cell based on an electrostatic layer-by-layer (LbL) technique on three-dimensional ordered macroporous (3DOM) gold electrode was described. A 3DOM gold electrode was synthesized electrochemically by an inverted colloidal crystal template technique. Then the macroporous gold electrodes were functionalized with Au nanoparticles (AuNPs) and enzyme, glucose dehydrogenase (GDH) or laccase. The (AuNPs/GDH)(n) multilayer modified macroporous gold electrode showed excellent bioelectrocatalytic activity towards glucose. The direct electroreduction towards oxygen was realized at (AuNPs/laccase)(n) films on 3DOM gold electrodes. The maximum power density of the cell with the macroporous film as matrix was 178muWcm(-2) at 226mV, which was 16 times larger than that of the biofuel cell with the flat electrode under the same condition. The proposed method is simple and would be applicable to enhance the power output of miniaturized biofuel cell.     

Enzymatic biofuel cell based on anode and cathode powered by ethanol

Enzymatic biofuel cell based on enzyme modified anode and cathode electrodes are both powered by ethanol and operate at ambient temperature is described. The anode of the presented biofuel cell was based on immobilized quino-hemoprotein-alcohol dehydrogenase (QH-ADH), while the cathode on co-immobilized alcohol oxidase (AOx) and microperoxidase (MP-8). Two enzymes AOx and MP-8 acted in the consecutive mode and were applied in the design of the biofuel cell cathode. The ability of QH-ADH to transfer electrons directly towards the carbon-based electrode and the ability of MP-8 to accept electrons directly from the same type of electrodes was exploited in this biofuel cell design. Direct electron transfer (DET) to/from enzymes was the basis for generating an electric potential between the anode and cathode. Application of immobilized enzymes and the harvesting of the same type of fuel at both electrodes (cathode and anode) avoided the compartmentization of enzymatic biofuel cell. The maximal open circuit potential of the biofuel cell was 240mV.     

Microfluidic biofuel cells: the influence of electrode diffusion layer on performance

Microfluidic biofuel cells exploit the lack of convective mixing at low Reynolds number to eliminate the need for a physical membrane to separate fuel from oxidant. This paper demonstrates how the length and spacing of electrodes within a microchannel, and thus thickness of the diffusion layer, affects the performance of a microfluidic biofuel cell. It was found that splitting a single electrode into two (or more) smaller electrodes and separating them by a distance equal to three times their length prevents the continuous increase in thickness of a diffusion layer. This change results in a 25% increase in maximum power density compared to a single electrode device with identical electroactive area. Furthermore, we found that the maximum current density of a microfluidic biofuel cell operated with different electrode configurations (i.e., length of cathode) closely matches that predicted by theory.     

On applicability of laccase as label in the mediated and mediatorless electroimmunoassay: effect of distance on the direct electron transfer between laccase and electrode

Applicability of laccase as enzyme-label has been investigated. It was shown that the property of laccase to catalyze the oxygen electroreduction at an electrode allows to develop a mediatorless and pseudoreagentless electro-enzyme-immunoassay (EEIA). In this case the electrode acts as an electron-donor substrate. When the bioelectrocatalytic reaction takes place, some electric charge is collected on the electrode. A method of determination of the electrode charge as well as the concentration of oxidized form of the mediator at the electrode surface has been elaborated. For this aim a technique of the measurement of current-surge was employed. Human immunoglobulin G and insulin were taken as model in this investigation. A back titration schemes without any mediator and in the presence of o-carboxybenzoylferrocene as a mediator was applied. The antibody carbon-black and the antigen glassy-carbon electrodes were used. The limits of detection were found to be 0.3 and 1.6 nM, respectively. The advantage of the mediatorless assay is that the charge leakage is imperceptible by open circuit for a long time and the accumulation of the charge occurs linearly with time. The charge accumulation for a long time allows to diminish the limit of detection. However, there is a limitation of the method. The direct electron transfer slows down with increasing the distance between the enzyme molecule and the electrode surface. This effect reduces the sensitivity of the method. The decrease of the electron transfer rate with distance has been estimated. Monolayer of hemoglobin dividing the laccase molecule from the electrode surface decreases the rate by four times. The electron transfer rate for the antibody electrode with associated antigen-laccase conjugate is less than that for the analogous electrode, covered with monolayer of covalently attached laccase, by 210 times. The current-surge peak was expected to decrease with distance by an equation of the form I = I0 exp[-r/r0]. The parameter r0 is equal to 2.2 +/- 0.8 nm. The possibility of the sensitivity increase in the mediatorless mode by 'wiring' through the multilayer film of immunoproteins immobilized on the electrode is discussed.     

Metabolic control analysis of an enzymatic biofuel cell

Metabolic control analysis (MCA) is an analytical technique that aims to quantify the distribution of control that enzymes exhibit over the steady‐state fluxes through a metabolic network. In an enzymatic biofuel cell, the flux of interest is the electrical current generated by the system. Regardless of transport limitations and other constraints, kinetic limitations can become potential bottlenecks in the operation of a biofuel cell. We have used an indirect approach to MCA to investigate a common osmium‐mediated glucose oxidase/laccase enzymatic biofuel cell. The results of the analysis show that the control of the electron flux strongly depends on the total mediator concentrations and the extent of polarization of the individual electrodes. The effect of varying oxygen concentrations is also examined, as oxygen is required for the cathode, but it participates in a non‐productive reaction at the anode. Under normal operating conditions the electrodes will be highly polarized and will both contain high mediator concentrations. This configuration will result in a dominant FCC at the anode, and the conditions that are needed for balanced flux control between the anode and cathode are explored. As increasingly complex bioelectrocatalytic systems and architectures are envisioned, MCA will be a valuable framework to facilitate their design and subsequent operation. Biotechnol. Bioeng. 2009;102: 1624–1635. © 2008 Wiley Periodicals, Inc.     

D-fructose detection based on the direct heterogeneous electron transfer reaction of fructose dehydrogenase adsorbed onto multi-walled carbon nanotubes synthesized on platinum electrode

Multi-walled carbon nanotubes (MWCNTs) were synthesized on platinum plate electrodes by the chemical vapor deposition (CVD) method. From the results of X-ray photoelectron spectroscopy and voltammetric investigation, the iron nanoparticles used as a catalyst for the MWCNT synthesis were enclosed with MWCNTs. The MWCNTs synthesized on the Pt plate (MWCNTs/Pt) electrode were immediately immersed into solutions of d-fructose dehydrogenase (FDH) to immobilize the enzyme onto the MWCNTs/Pt electrode surfaces. After the FDH was immobilized onto the MWCNTs/Pt electrode, a well-defined catalytic oxidation current based on FDH was observed from ca. -0.15V (versus Ag/AgCl/sat'd KCl), which was close to the redox potential of heme c as a prosthetic group of FDH. From an analysis of a plot of the catalytic current versus substrate, the calibration range for the fructose concentration was up to ca. 40mmoldm(-3), and the apparent Michaelis-Menten constant was evaluated to be 11+/-1mmoldm(-3).     

Enzymatic fuel cells: integrating flow-through anode and air-breathing cathode into a membrane-less biofuel cell design

One of the key goals of enzymatic biofuel cells research has been the development of a fully enzymatic biofuel cell that operates under a continuous flow-through regime. Here, we present our work on achieving this task. Two NAD(+)-dependent dehydrogenase enzymes; malate dehydrogenase (MDH) and alcohol dehydrogenase (ADH) were independently coupled with poly-methylene green (poly-MG) catalyst for biofuel cell anode fabrication. A fungal laccase that catalyzes oxygen reduction via direct electron transfer (DET) was used as an air-breathing cathode. This completes a fully enzymatic biofuel cell that operates in a flow-through mode of fuel supply polarized against an air-breathing bio-cathode. The combined, enzymatic, MDH-laccase biofuel cell operated with an open circuit voltage (OCV) of 0.584 V, whereas the ADH-laccase biofuel cell sustained an OCV of 0.618 V. Maximum volumetric power densities approaching 20 μW cm(-3) are reported, and characterization criteria that will aid in future optimization are discussed.     

Incorporation of Indium Tin Oxide Nanoparticles in PEMFC Electrodes

Carbon materials suffer from corrosion at the cathode of polymer electrolyte membrane fuel cells (PEMFCs). In the presence of water, carbon support materials are oxidized to carbon dioxide even at low potentials. Hence, nowadays it is very fashionable to look for alternative support materials, like oxides or conductive polymers. To gain the maximum performance for a new material one should also consider an appropriate electrode structure. This study shows the results for the incorporation of nanosized alternative support materials into advanced electrode architectures. Commercially available indium tin oxide (ITO) nanoparticles (<50 nm) are used as support for Pt nanoparticles in combination with Nafion‐coated multi‐walled carbon nanotubes (MWCNTs) on the cathode side of a PEMFC. The MWCNTs promote a high electronic conductivity and help to form a porous network, which could accommodate the Pt/ITO nanoparticles. The microscopic investigations show a homogeneous electrode structure composed of Pt/ITO and MWCNT/Nafion multilayer. Single cell measurements show a maximum power density of 73 mW cm^?2 and a Pt utilization of 1468 mW mg_Pt^?1 for the cathode. The performance data and the Pt utilization are comparable to a standard Pt/carbon black electrode possessing the same Pt loading in the electrode. Beside this, it is shown for the first time that ITO serves as support material under real fuel cell conditions.     

Construction and direct electrochemistry of orientation controlled laccase electrode

A laccase has multiple redox centres. Chemisorption of laccases on a gold electrode through a polypeptide tag introduced at the protein surface provides an isotropic orientation of laccases on the Au surface, which allows the orientation dependent study of the direct electrochemistry of laccase. In this paper, using genetic engineering technology, two forms of recombinant laccase which has Cys-6×His tag at the N or C terminus were generated. Via the Au-S linkage, the recombinant laccase was assembled orientationally on gold electrode. A direct electron transfer and a bioelectrocatalytic activity toward oxygen reduction were observed on the two orientation controlled laccase electrodes, but their electrochemical behaviors were found to be quite different. The orientation of laccase on the gold electrode affects both the electron transfer pathway and the electron transfer efficiency of O₂ reduction. The present study is helpful not only to the in-depth understanding of the direct electrochemistry of laccase, but also to the development of laccase-based biofuel cells.     

High Active Material Loading in All‐Solid‐State Battery Electrode via Particle Size Optimization

Low active material loading in the composite electrode of all‐solid‐state batteries (SSBs) is one of the main reasons for the low energy density in current SSBs. In this work, it is demonstrated with both modeling and experiments that in the regime of high cathode loading, the utilization of cathode material in the solid‐state composite is highly dependent on the particle size ratio of the cathode to the solid‐state conductor. The modeling, confirmed by experimental data, shows that higher cathode loading and therefore an increased energy density can be achieved by increasing the ratio of the cathode to conductor particle size. These results are consistent with ionic percolation being the limiting factor in cold‐pressed solid‐state cathode materials and provide specific guidelines on how to improve the energy density of composite cathodes for solid‐state batteries. By reducing solid electrolyte particle size and increasing the cathode active material particle size, over 50 vol% cathode active material loading with high cathode utilization is able to be experimentally achieved, demonstrating that a commercially‐relevant, energy‐dense cathode composite is achievable through simple mixing and pressing method.     

Direct electron transfer catalysed by enzymes: application for biosensor development

The ability to catalyse an electrode reaction via direct (mediatorless) electron transfer has been demonstrated for a number of redox enzymes. In the case of mediatorless electron transfer, the electron is transferred directly from the electrode to the substrate molecule via the active site of the enzyme, or vice versa. The electron itself is the second substrate for the reaction. An important point characterizing bioelectrocatalysis is the catalytic removal of the reaction over-voltage. Therefore the enzyme attached to the electrode is able to catalyse electrode reaction and forms a 'molecular transducer'. The substrate can be detected by potentiometric measurement of the removal of reaction over-voltage. The enzyme laccase is able to catalyse the reaction of oxygen electroreduction. Therefore a laccase molecular layer attached to the electrode surface forms an oxygen transducer. The formation of the layer results in a change of the electrocatalytic feature of the electrode. Laccase label coupled with either ligand or receptor allows the detection of ligand-receptor complex formation/dissociation on the electrode surface. The detection is virtually reagentless. The substrates for the reaction are molecular oxygen and the electron itself. Numerous reagentless immunosensors of different formats (competitive, displacement and sandwich) have been developed, as well as the reagentless detection system for immunofiltration/immunochromatography.     

Electrically Contacted Bienzyme‐Functionalized Mesoporous Carbon Nanoparticle Electrodes: Applications for the Development of Dual Amperometric Biosensors and Multifuel‐Driven Biofuel Cells

The capping of electron relay units in mesoporous carbon nanoparticles (MPC NPs) by crosslinking of different enzymes on MPC NPs matrices leads to integrated electrically contacted bienzyme electrodes acting as dual biosensors or as functional bienzyme anodes and cathodes for biofuel cells. The capping of ferrocene methanol and methylene blue in MPC NPs by the crosslinking of glucose oxidase (GOx) and horseradish peroxidase (HRP) yields a functional sensing electrode for both glucose and H₂O₂, which also acts as a bienzyme cascaded system for the indirect detection of glucose. A MPC NP matrix, loaded with ferrocene methanol and capped by GOx/lactate oxidase (LOx), is implemented for the oxidation and detection of both glucose and lactate. Similarly, MPC NPs, loaded with 2,2′‐azino‐bis(3‐ethylbenzo­thiazoline‐6‐sulphonic acid), are capped with bilirubin oxidase (BOD) and catalase (Cat), to yield a bienzyme O₂ reduction cathode. A biofuel cell that uses the bienzyme GOx/LOx anode and the BOD/Cat cathode, glucose and/or lactate as fuels, and O₂ and/or H₂O₂ as oxidizers is assembled, revealing a power efficiency of ≈90 μW cm^?2 in the presence of the two fuels. The study demonstrates that multienzyme MPC NP electrodes may improve the performance of biofuel cells by oxidizing mixtures of fuels in biomass.